KR20170064540A - Target sequence detection by nanopore sensing of synthetic probes - Google Patents

Abstract

Methods and compositions are disclosed herein for detecting one or more specific sequences of a polynucleotide in solution using a nanopore. In some embodiments, methods and compositions for identifying polynucleotides in a sample or detecting a target sequence of a polynucleotide are disclosed herein.

Description

[0001] TARGET SEQUENCE DETECTION BY NANOPORE SENSING OF SYNTHETIC PROBES [0002]

relation Cross reference to application

This application claims the benefit of U.S. Provisional Patent Application No. 62 / 056,378, filed September 26, 2014, the disclosure of which is incorporated herein by reference.

Field of the Invention

The present invention relates to a method and a composition for detecting a target sequence using a nanopore device.

The detection, localization, and copy number determination of specific sequence regions within the stretch of nucleic acids, also referred to as "target sequence detection ", applies to the life science and technology, medical, agricultural and forensic fields as well as other fields. Detection of genes and their modifications, sequences, positions, or numbers is important in the development of molecular diagnostics in medicine. DNA microarrays, PCR, Southern blot, and FISH (Fluorescent in situ Hybridization) are all methods that can be used to perform or assist in target sequence detection. These methods are slow, labor intensive, and have limited accuracy and resolution. More recent methods, such as real-time PCR and next generation sequencing (NGS) techniques, have improved throughput, but still do not have sufficient resolution for many applications.

Solid state nanopores have been shown to detect molecules by applying a voltage across the pores and measuring the current impedance as the molecules pass through the nanopore. The overall efficiency of any given nanopore device depends on its ability to accurately and reliably measure the AC impedance and distinguish it from other types of passing molecules. The experiments presented in the literature demonstrate the detection of both DNA and RNA strands passing through the pores, and synthetic molecules that hybridize to specific sequences in them. However, they could not be used to create high throughput and reliable nanopore devices for detecting probes in specific DNA or RNA sequences. Probes developed so far are insufficient for reliable sequence detection. Therefore, there is a need for a set of probe and probe complexes that are capable of sequence-specifically binding for detection in nanopores.

There is provided herein a method of detecting a polynucleotide comprising a target sequence in a sample wherein said sample is specific for said polynucleotide comprising said target sequence and said sample under conditions that promote binding of said probe to said target sequence Contacting probe under conditions that promote binding of said probe to said target sequence to form a polynucleotide-probe complex; Loading the sample into a first chamber of a nanopore device, wherein the nanopore device includes at least one nanopore and at least the first chamber and the second chamber, Wherein the nanopore device further comprises a voltage associated with each of the at least one nanofourous and a voltage that is independently controlled across each of the at least one nanofoure, Wherein the sensor is configured to identify an object passing through at least one nanopore and wherein the translocation of the polynucleotide-probe complex through the at least one nanopore is detectable with respect to the polynucleotide-probe complex Providing a signal; And observing the detectable signal to determine the presence or absence of the polynucleotide-probe complex in the sample by detecting the polynucleotide comprising the target sequence. In one embodiment, the method further comprises generating a voltage potential through the at least one nanopore, wherein the voltage potential is generated by generating a force on the polynucleotide-probe complex to form the at least one nanopore Probe complex, wherein the polynucleotide-probe complex is displaced through the at least one nanopore to generate the detectable signal.

In some embodiments, the polynucleotide is DNA or RNA. In one embodiment, the detectable signal is an electrical signal. In one embodiment, the detectable signal is an optical signal. In one embodiment, the probe comprises a molecule selected from the group consisting of a protein, a peptide, a nucleic acid, TALEN, CRISPR, a peptide nucleic acid, or a compound. In one embodiment, the probe is selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), DNA / RNA hybrid, polypeptide, or any chemically derivatized polymer Includes selected molecules.

In one embodiment, the probe comprises a PNA molecule bound to a second molecule configured to facilitate detection of a probe bound to the polynucleotide in a potential across the at least one nanopore. In a further embodiment, the second molecule is PEG. In a further embodiment, the PEG has a molecular weight of at least 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, or 10 kDa.

In one embodiment, the method for detecting a polynucleotide comprising a target sequence in a sample further comprises applying to the sample a condition suspected of altering the binding interaction between the probe and the target sequence. In a further embodiment, the conditions are selected from the group consisting of removal of the probe in the sample, addition of an agent competing with the probe for binding to the target sequence, and a change in initial pH, salt, or temperature conditions.

In one embodiment, the polynucleotide comprises a chemical modification configured to modify the binding of the polynucleotide to the probe. In a further embodiment, the chemical modification is selected from the group consisting of biotinylation, acetylation, methylation, summation, glycosylation, phosphorylation and oxidation.

In one embodiment, the probe comprises a chemical modification coupled to the probe via a cleavable bond. In one embodiment, the probe is a probe of a polynucleotide through covalent bonds, hydrogen bonds, ionic bonds, metal bonds, van der Waals forces, hydrophobic interactions, or planar stacking interactions. Interact with the target sequence. In one embodiment, the method for detecting a polynucleotide comprising a target sequence in a sample further comprises contacting the sample with at least one detectable label capable of binding to the probe or polynucleotide-probe complex. In one embodiment, the polynucleotide comprises at least two target sequences.

In one embodiment, the nanopore is about 1 nm to about 100 nm in diameter and 1 nm to about 100 nm in length, and each chamber includes an electrode. In one embodiment, the nanopore device comprises at least two nanopores configured to simultaneously control the movement of the polynucleotide in two nanopores. In one embodiment, the method for detecting a polynucleotide comprising a target sequence in a sample comprises detecting the presence of the polynucleotide in the sample by passing the probe-binding moiety through the nanopore and inverting the movement of the polynucleotide through the nanopore, Reversing the independently controlled voltage after the initial detection of the polynucleotide-probe complex, thereby again confirming the presence or absence of the polynucleotide-probe complex.

In one embodiment, the nanopore device comprises two nanopores, the polynucleotides being simultaneously located in both of the two nanopores. In a further embodiment, the method for detecting a polynucleotide comprising a target sequence in a sample comprises the steps of: determining the concentration of the polynucleotide in the sample, And adjusting the magnitude and / or direction of the voltage.

Also provided herein is a method for detecting a polynucleotide or polynucleotide sequence in a sample, said method comprising contacting said sample with a first probe and a second probe, wherein said first probe comprises a first probe and a second probe, 1 probe under conditions that facilitate binding of the second probe to the second target sequence, wherein the second probe specifically binds to the first target sequence of the polynucleotide under conditions that promote binding of the second probe to the second target sequence, Lt; RTI ID = 0.0 > of: < / RTI > The sample is configured to be simultaneously bonded to the first and second probes when the first and second probes are sufficiently close to each other under conditions that promote binding of the third molecule to the first probe and the second probe, To form a fusion complex comprising the polynucleotide, the first probe, the second probe, and the third molecule; Loading the sample into a first chamber of a nanopore device, wherein the nanopore device includes at least one nanopore and at least the first chamber and the second chamber, Wherein the nanopore device further comprises a sensor associated with each of the at least one nanopore and a voltage potential controlled across each of the at least one nanopore, Wherein the sensor is configured to identify an object passing through the at least one nanopore, wherein the fusion complex potential through the at least one nanopore is indicative of a detectable signal associated with the fusion complex; And observing the detectable signal to determine the presence or absence of the fusion complex in the sample.

In one embodiment, the polynucleotide is DNA or RNA. In one embodiment, the detectable signal is an electrical signal. In one embodiment, the detectable signal is an optical signal. In one embodiment, the sufficient proximity is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or less than 500 nucleotides. In one embodiment, the third molecule comprises a PEG or an antibody.

In one embodiment, the third molecule and the first and second probes are bound to ssDNA, and the ssDNA linked to the third molecule comprises a region complementary to a region of ssDNA linked to the first probe, 2 < / RTI > probe. In one embodiment, a method of detecting a polynucleotide or polynucleotide sequence in a sample further comprises contacting the sample with a third molecule or one or more detectable labels capable of binding to the fusion complex.

Also provided herein is a kit comprising a first probe, a second probe, and a third molecule, wherein the first probe is configured to bind to a first target sequence on the target polynucleotide and the second probe is configured to bind on the target polynucleotide Wherein the third molecule is configured to bind to a first probe and a second probe when the first and second probes are bound to the polynucleotide in the first and second target sequences, Thereby positioning the first and second probes sufficiently close to allow simultaneous bonding of the third molecule to the first and second probes.

In one embodiment, the first probe and the second probe are selected from the group consisting of a protein, a peptide, a nucleic acid, a TALEN, a CRISPR, a peptide nucleic acid, or a compound. In one embodiment, the third molecule comprises a PEG or an antibody. In one embodiment, the third molecule comprises a modification to modify the binding affinity for the probe.

Also provided herein are nanopore devices comprising at least two chambers and nanopores, wherein the device comprises a modified PNA probe coupled to the polynucleotide in the nanopore.

A dual-pore, dual-amplifier device for detecting charged polymers through two pores is also provided herein, the device comprising an upper chamber, an intermediate chamber and a lower chamber, wherein the first pore comprises an upper chamber and a middle chamber, And a second pore connects the intermediate chamber and the lower chamber, wherein the device comprises a modified PNA probe coupled to the polynucleotide in the first or second pore.

In one embodiment, the device is configured to simultaneously control the movement of the charged polymer through both the first pore and the second pore. In one embodiment, the modified PNA probe is bound to at least one PEG molecule. In one embodiment, the device further comprises a power supply configured to provide a first voltage between the upper chamber and the intermediate chamber and to provide a second voltage between the intermediate chamber and the lower chamber, wherein each voltage is independently adjusted Wherein the intermediate chamber is connected to a common ground for two voltages and the device provides a dual-amplifier electronics configured for independent voltage control and current measurement in each pore, and the two voltages may be of different sizes Wherein the first and second pores are configured such that the charged polymer can move simultaneously across the two pores in both directions and in a controlled manner.

The drawings, which are shown by way of example only and not by way of limitation, are provided as examples of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the detection of a target molecule bound to a probe modified in pairs in a nanopore as an embodiment of the method disclosed herein.
Figure 2 shows the effect of probe binding to a target molecule on the electrical signal generated when the complex is displaced through the nanopore.
3A and 3B each show two probes bound to a polynucleotide in each target sequence and a third bridging to facilitate probe detection in the nanopore when the two probes are bound to a scaffold ) Molecule (e. G., An antibody).
Figure 4 shows two probes bound to the polynucleotide in each target sequence, the third bridging molecule PEG and a complementary ssDNA linker to enable probe detection when the two probes are sufficiently close to the scaffold Lt; / RTI >
Figure 5 shows two probes coupled to the polynucleotides of each target sequence sufficiently close enough to detect an optical signal generated through, for example, Poster Resonance Energy Transport (FRET) due to proximity.
6A is a schematic diagram of a system combining a nanopore device and an epifluorescence microscope to enable detection of a fluorescent monosorbable binder. 6B is an illustration of the detector as it passes through two nanopore devices in a plane. Figure 6C shows the change in current amplitude and the corresponding fluorescence signal as the scaffold passes through the nanopore.
Figure 7 illustrates the coupling of a probe with a cleavable group (e.g., a fluorophore) to aid in detection.
Figure 8 shows the multiple capabilities of the technique by including probes of different sizes, each binding to a unique target sequence in a target containing molecule. In this illustration, double-stranded DNA is a polynucleotide with a target sequence and a number of other DNA-binding probes that bind to the target sequence desired to be detected.
Figure 9A shows a modified PNA ligand to increase the ligand charge to facilitate detection by the nanopore. Figure 9B shows an example in which double-stranded DNA is used as a target-containing polymer and a number of other DNA binding probes that bind to a target sequence desired to be detected.
Figure 10 shows a number of distinct sequence-specific probes that are coupled to DNA as they pass across the nanopore to allow multiplexed detection.
Figures 11A-C show representative current signatures and populations of potentials of molecules through nanopores and nanopores. 11A, a solid state pore and a voltage path are shown. Figure 11B shows the current interruption and residence time of the molecules passing through the nanopore. Figure 11C shows a characteristic population of molecules passing through a nanopore based on residence time and average current amplitude.
Figure 12A shows an example of the use of a PNA probe conjugated to biotin to form complexes with larger neutravidin molecules to allow detection of the sequence in the DNA scaffold. Figure 12B shows the binding site for the PNA probe in the DNA scaffold.
Figure 13 shows the potential of PNA-biotin conjugated to unbound DNA, free neutravidin, and DNA and neutravidin. The current signature (y-axis current, x-axis time) obtained when the molecule is displaced through the nanopore under an applied voltage for each complex also appears.
DNA / PNA / Nutravidin complexes are more detectable than other background event types (e. G., Unconjugated DNA alone, and neurotavidin alone), and detectable PNA probes (i.e., DNA / PNA / Lt; / RTI > complex).
Figure 14A shows the time and average conductivity shift due to dislocation through nanopores in DNA (circle) complexed with three groups, DNA alone (x), neutravidin single (square), and biotin probes attached to neutravidin ≪ / RTI > Figure 14B shows a histogram of the residence time probabilities associated with each of the three groups described above. FIG. 14C shows a sample containing DNA alone (lane 2), DNA, PNA with three biotin sites for binding to neutravidin and lupine (lane 3), DNA, seven biotin moieties for binding to neutravidin (Lane 3), DNA, PNA with 16 biotinyl moieties for binding to neutravidin, and a sample containing neutravidin (lane 3), and DNA, (Lane 3) containing PNA with 36 biotinyl moieties for conjugation, and neutravisin for binding.
Figure 15 shows a plot of probe binding sites on a DNA scaffold, wherein the probe is a VspR protein.
Figure 16A shows a diagram of representative current signatures associated with unbound DNA molecules passing through the nanopore, and a single molecule passing through the nanopore. Figure 16B shows a representative current signature associated with the VspR-bound DNA molecule passing through the nanopore, and its nanopore pass.
Figure 17 shows at least ten representative current decay events consistent with the VspR-coupled scaffold through the pores.
Figure 18A shows a PNA-PEG probe attached to a target sequence in a dsDNA molecule. Figure 18B shows the results of a gel migration assay using the following samples: DNA alone (lane 1), DNA / PNA (lane 2), DNA / PNA-PEG (lane 3) PEG (20 kDa) (lane 4). Figure 18C shows the results of a gel migration assay using the following samples: a DNA marker (lane 1), a random DNA sequence incubated with a PNA probe (lane 2), a single miss in the target sequence incubated with the PNA probe DNA with a match (lane 3), and DNA with a target sequence mixed with the corresponding PNA probe specific for the target sequence (lane 4).
19A shows a representative current signature event in which each current signature is displaced through a nanopore under an applied voltage as the molecule shown below. Figure 19B is characterized by the duration and average conductivity shift due to dislocations across the nanopore in three groups: DNA / bisPNA (square), DNA / bisPNA-PEG 5kDa (circle), and DNA / bisPNA-PEG 10kDa Of the event. Figure 19C shows a histogram of average conductivity shift probabilities associated with each of the three groups described above. Figure 19D shows a histogram of event duration probabilities associated with each of the three groups described above.
Figure 20A shows representative event signatures associated with the potential of PNA-PEG probes bound to DNA molecules. Figure 20B shows the mean conductivity shift versus time plot for events recorded respectively in nanopores of samples containing bacterial DNA and PNA-PEG probes. Figures 20C and 20D show corresponding histograms for characterizing these events detected by the average conductivity shift and duration of each event. Figure 20E shows the results of the gel migration assay as follows: 100 bp ladder (lane 1), 300 bp DNA with wild type cftr sequence incubated with PNA-PEG probe (lane 2), and incubation with PNA- 300 bp DNA with the cftr? F508 sequence (lane 3).
Figure 21A are the determination of the gel moves black, lane 1 contains the S. mitis bacterial DNA is bisPNA PEG-unbound, and lane 2 is a part-includes a S. mitis DNA binding is specific bisPNA-PEG . Figure 21B shows the scatter plot of the mean conductivity shift (dG) on the ordinate vs. the duration on the abscissa for all events recorded in two subsequent experiments. The first sample contained bacterial DNA (DNA / bisPNA-PEG) with a PEG-modified PNA probe. The second sample contained bacterial DNA alone.
Some or all of the figures are schematic diagrams for illustration; Accordingly, they do not necessarily indicate the actual relative sizes or locations of the illustrated elements. The drawings are provided for the purpose of illustrating one or more embodiments having a clear understanding without limiting the scope or meaning of the following claims.

Throughout this application, this specification represents various embodiments of the present nutrients, compositions, and methods. The various embodiments described are meant to provide various exemplary embodiments and should not be construed as an explanation of alternative species. Rather, it should be understood that the description of various embodiments provided herein may be duplicated. The embodiments discussed herein are illustrative only and are not meant to limit the scope of the present invention.

In addition, throughout this disclosure, various publications, patents, and published patent specifications are hereby incorporated by reference. The disclosures of these publications, patents, and published patent specifications are incorporated herein by reference in order to more fully describe the state of the art to which the present invention is directed.

The singular forms " a ", "an ", and" an ", as used in the specification and claims, include a plurality of referents unless the context clearly dictates otherwise. For example, the term "electrode" includes a plurality of electrodes and includes the mixture.

The term "comprising " as used herein is intended to mean that the device and method include the recited element or step, but do not preclude others. "Essentially composed" means excluding other elements or steps having any essential significance to the combination when used to define the device and method. "Comprising" means excluding another element or step. Embodiments defined by each of these transition terms are within the scope of the present invention.

All nomenclature, e.g., distance, size, temperature, time, voltage, and concentration, including ranges, are approximations that are intended to include conventional experimental changes in the measurement of parameters, . It should be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about ". Also, while not always expressly stated, it is to be understood that the components described herein are illustrative only and that equivalents of those are known in the art.

The term "nanopore" (or "pore") refers to a single nano-scale opening of a membrane separating two volumes as used herein. The pores can be, for example, protein channels embedded in a lipid bilayer membrane or can be formed by drilling, etching or by using a thin solid state substrate, such as silicon nitride or silicon dioxide, Or graphene, or a combination of these or other materials. Geometrically, the diameter of the pores is between 0.1 nm and 1 micron; The length of the pores is determined by the film thickness, which may be less than nanometers or up to 1 micron or more. For membranes thicker than a few hundred nanometers, nanopores may be referred to as "nanochannels ".

As used herein, the term "nanopore device" refers to a device in which one or more nanopores and a circuit for sensing single molecule events are combined (in parallel or in series). Specifically, a nanopore instrument uses a sensitive voltage-clamp amplifier to measure a current through a pore while applying a specific voltage across the pores or pores. When a single-charged molecule, such as double-stranded DNA (dsDNA), is captured and driven through the pore by electrophoresis, the measured current shifts, which is a function of the capture event (i.e. the potential of the molecule through the nanopore, Capture of molecules in the pore), and the amount of shift of the event (current amplitude) and duration are used to characterize the molecules captured in the nanopore. After recording many events during the experiment, the event distribution is analyzed and the corresponding molecules are characterized according to the shift amount (i.e., current signature). Thus, nanopores provide a simple, label-free, purely electrical, single-molecule method for detecting biomolecules.

As used herein, the term "event" refers to the potential of a molecule or molecular complex detectable through a nanopore and the measurement associated therewith. The event may be defined by current, duration, and / or other characteristics of the detection of the molecule in the nanopore. A plurality of events with similar characteristics represent a group of molecules or complexes having the same or similar characteristics (e.g., bulk, charge).

Molecular detection

The present disclosure provides methods and systems for the detection and quantification of molecules. The method and system can also be configured to measure the affinity of a probe that binds to a target molecule. Further, such detection, quantification, and measurement can be performed in a multiplexed manner, which greatly increases efficiency.

Figure 1 provides an illustration of one embodiment of the disclosed method and system. More specifically, the system includes a target containing molecule 102 containing a target motif 101 that it desires to be detected or quantified. The probe 103 can bind to the specific binding motif 101 on the target-containing molecule 102. Additional molecules may be added to aid in the detection of probes 107 present on the target containing polynucleotide.

Therefore, when all are present in the solution, the probe 103 binds to the target motif through specific recognition of the probe to the target motif 101. [ Such binding results in the formation of a complex comprising the probe and the target sequence.

The formed composite 101/103 or 101/103/107 includes two pores 105 and 106 separating the internal space of the device into three volumes and a sensor adjacent to the pore configured to identify an object passing through the pore Lt; RTI ID = 0.0 > 104 < / RTI > This embodiment is a dual nanopore device with two nanopores in series. In some embodiments, the nanopore device is capable of generating a controlled voltage across the nanopore (in some embodiments, the voltage can be independently controlled and clamped), along with circuitry for measuring current flow across the nanopore And electronic components for delivery. The voltage can be adjusted to move polynucleotides from one volume to another across the pores in a controlled manner. The poly-nucleic acid is modified to be charged or charged and the applied potential or voltage differential across the pore facilitates movement of the charged scaffold through the application of electrostatic force on the charged molecule exposed to the voltage field Respectively. Figure 1 shows a dual nanopore device, but the principles described above can be applied to other embodiments of the invention using a single nanopore device. Unless otherwise specified, references to pore or nanopore or nanopore devices are intended to include single, dual or multi-pore devices within the scope of the present invention.

When a sample comprising the formed complex is loaded into the nanopore, the nanopore can be configured such that the target containing molecule passes through the pore. When the target motif is within the pore or adjacent to the pore, the binding state of the target motif can be detected by the sensor.

The "binding state" of the target motif refers to whether the binding motif is occupied by the probe, as used herein. Essentially, the bonded state is bonded or unbound. Also, (i) the target motif is liberated and not bound to the probe (see 201 and 204 in FIG. 2) or (ii) the target motif is bound to the probe (see 202 and 205 in FIG. 2). Additionally, probes of different size or with different probe binding sites may be used to provide additional current profiles (see, e.g., 203 and 206 in FIG. 2) such that one or more target sequences can be detected in one target containing molecule .

Detection of the binding state of the target motif can be performed in various ways. In one embodiment, different sizes of target motifs in each state (i.e., occupied or unoccupied) cause different magnitudes to generate different currents across the pores as the target motif passes through the pores. In this regard, a separate sensor is not required for detection, since an electrode connected to a power source and capable of detecting current can provide a sensing function. Therefore, the two electrodes can act as "sensors ".

In some embodiments, an agent (e. G., 107 in Figure 1) is added to the complex to add detection. This agent can bind to the probe or polynucleotide / probe complex. In one embodiment, the agonist includes charge, either negative or positive, to facilitate detection. In another embodiment, the agent adds a size to facilitate detection. In another embodiment, the agent comprises a detectable label, such as a fluorescent moiety.

In this context, identification of the bound state (ii) indicates that the target sequence in the target containing molecule forms a complex with the probe. In other words, the target sequence is detected.

In another embodiment, the bound molecules are spaced apart to individually detect bound molecules by impedance changes, and each bound molecule provides an impedance value that is not masked by the adjacent bound molecule.

In one embodiment, the bound probe is separated by a distance of at least 1 nm (i.e., approximately 3 bp relative to the nucleic acid-based polynucleotide). In another embodiment, the bound probe is separated by a distance of at least 10 nm (i.e., approximately 33 bp relative to the nucleic acid-based polynucleotide). In yet another embodiment, the bound probe is separated by a distance of at least 100 nm (i.e., approximately 333 bp relative to the nucleic acid-based polynucleotide). In yet another embodiment, the bound probe is separated by a distance of at least 500 nm (i.e., approximately 1666 bp relative to the nucleic acid-based polynucleotide).

In some embodiments, the method further comprises the step of having two independent probes capable of binding to a third molecule when they are sufficiently close to each other when bound to the polynucleotide. This binding of the third molecule provides different potential current signatures, providing evidence that two independent probes are in close proximity.

The mechanism for determining whether a probe binds in close proximity allows the inventors to detect single base pair mismatches, detect alleles with a single nucleotide polymorphism (or single nucleotide mutation), and longer probes, Enabling the use of short probes acting as markers to establish a unique position. In one embodiment, the inventors use two probes combined to determine whether a particular sequence is associated with a particular target gene. In another embodiment, the method is used to determine a structural rearrangement by using a probe as a marker for a region of the genome. In another embodiment, the inventors use two probes combined to determine whether a particular sequence is chemically modified (e. G., Genetically, e.g. methylated, hydroxymethylated) and associated with a particular target gene .

In one embodiment, the third molecule is an antibody that binds only to probes 305 and 306, if at least two probes are bound to polynucleotide 304 and are fairly close to each other (0.01 nm to 50 nm) (Figure 3A). In yet another embodiment, half of the epitope for antibody 301 is linked to the respective probe molecules 302 and 303 (via covalent attachment, ionic, H-bonding, etc.) Dependent on the two epitopes located close together, indicating that the probes are close enough together (Figure 3B). In one embodiment, each probe is a PNA molecule, and each PNA molecule comprises or is attached to a segment of the binding epitope for the antibody (attachment by covalent bonds, ionic, H-bonding, etc.). In this embodiment, the partial epitope should be close enough for the antibody to bind to form a complex with the polynucleotide.

In another embodiment, the PEG molecule 310 is used to bind the two probes 302 and 303 very closely as a third molecule and binds to the polynucleotide 304. In some embodiments, the PEG is modified to provide sufficient bulk, charge, or other features that permit a unique signature when present (FIG. 4). In some embodiments, the PEG is modified to increase the binding affinity for the proximal probe. This binding modification on PEG may be, for example, a ssDNA molecule complementary to free single-stranded DNA (ssDNA) attached to each probe at each end of the PEG. In one embodiment, the energy barrier required for PEG binding to the probe is met only when two ssDNA oligos are attached to their complementary sequences attached to the probe (Figure 4). Thus, probes at a distance over which PEG spans are detected in nanopores through the detection of PEG / probe / polynucleotide complexes, while distant ones are not. Those skilled in the art will recognize that ssDNA can be replaced by synthetic nucleic acid analogs, such as PNA or RNA.

In some embodiments, two independent probes are modified to allow detection when they are bound to the polynucleotide in close proximity. In one embodiment, the modification to the probe is such that the probe is bound to the polynucleotide in very close proximity, as distinguished from the current signature when the single probe / polynucleotide complex passes through the nanopore without being very close to the second probe And includes ion changes in the probe to change the current signature as it passes through the nanopore. In one embodiment, adding a positive charge to two probes (as by labeling the probe with, for example, 2-hydroxyethyl thiosulfonate (MTSET)), as opposed to binding to a polynucleotide farther away, Is spatially close enough along the polynucleotide and provides a different potential current signature when the effect of charge is added.

In some embodiments, the method further comprises the step of using a probe that is long enough to allow binding to only one unique sequence in the target population, but has the ability to not bind to the target site when only a single base pair mismatch is present . This is possible when using PNA probes. (Strand-Invasion of Extended, Mixed-Sequence B-DNA by? PNAs, G. He, D. Ly et al., J Am Chem Soc. 2009 September 2; 131 (34): 12088-12090. / ja900228j), the 20 bp gamma-PNA probe can efficiently bind to a perfectly matched target sequence, but the binding is abolished if the target sequence and probe sequence differ even by a single base. Considering the human genome containing 3.1 billion bases, the 20 base pair sequence is likely to occur randomly 0.003 times. Thus, the 20 base pair probes designed to bind to the specific sequence under investigation are very unlikely to bind to unwanted sites and provide false positives. The examples contained in Figures 19C and 21 show that the PNA and PNA-PEG probes selectively bind only to complementary sequences.

In some embodiments, the method further comprises the step of having two independent probes comprising elements that emit a detectable signal when the two probes are sufficiently close to the polynucleotide to be attached. In one embodiment, each probe is labeled with a fluorophore (see, e.g., Fig. 5 (315, 316)). The emission spectrum is detected by the detector 317 when the probe is close enough to generate a detectable signal. In one embodiment, the two probes are labeled with different colored fluorophore ends. When the probe is in close proximity, colors that can be detected with an external sensor, such as a camera or a microscope, will be imaged (or blended to provide a new color) together, which is evidence that the two probes are spatially close together. In a related embodiment, FRET (or BRET) type detection is used to determine if two probes are close enough so that, for example, one fluorescently labeled probe affects the other's energy release spectrum .

In some embodiments, the detectable label is a fluorophore. To detect the fluorescence end, a planar nanopore device with a glass cover can be combined with an epifluorescence microscope to enable double current amplitude and fluorescence signal detection. Figure 6A shows how such a device can be used to detect added fluorescence labeling. The nanopore device is placed under the objective lens of the epifluorescence microscope. When a nanopore measurement is performed, the microscope continues to image the nanopore region. The nanopore region is irradiated by a broad excitation source that is filtered to pass only the wavelength corresponding to the excitation spectrum of the fluorophore. A dichroic filter reflects all other wavelengths while permitting selective transmission of wavelengths corresponding to the emission spectrum of the fluorophore. When the fluorescent moiety-modified binding molecule passes through the nanopore, the fluorophore absorbs the excitation spectrum and re-emits the emission spectrum. The emission filter in front of the detector ensures that only wavelengths corresponding to the emission spectrum of the fluorescence end are detected. Therefore, the detector will only have a signal when the fluorescence end passes through the nanopore. FIG. 6B shows a top view of a nanopore device viewed under a microscope during the emission of the fluorescent tip. Figure 6C shows whether the detection of the fluorophore can be used with the signal of the nanopore. The use of two signals improves the reliability of detection of biomolecules.

In some aspects, the method further comprises using a probe having an object that is detected by the sensor but is attached to the probe using a severable linker. Thus, a probe set that can be distinguished from one another in a nanopore is bound to a target containing polynucleotide. When the probe set is detected in the nanopore, a new set of probes with objects to be cut and also with detectable objects is added (Fig. 7). The add / cut / wash cycles can be continued until all the sequence information is extracted from the captured target molecule. Examples of molecules that aid in probe detection are described above. Examples of cleavable linkers include, but are not limited to, a reducing cleavable linker (a disulfide linker cleaved by TCEP), an acid cleavable linker (hydrazone / hydrazide linkage), an amino acid sequence cleaved by a protease, an endonuclease Enzymes), base-cleavable linkers, or light-cleavable linkers [Leriche, Geoffray, Louise Chisholm, and Alain Wagner. "Cleavable linkers in chemical biology." Bioorganic & medicinal chemistry 20, no. 2 (2012): 571-582].

Target motif

For nucleic acids and polypeptides to which the target sequence detection method is applied, the target binding motif may be a nucleotide or peptide sequence recognizable by the probe molecule. The target motif may be chemically modified (e. G., Methylated) or occupied by another molecule (e. G., An activator or inhibitor) and the state of the target motif may be described, depending on the nature of the probe. In some embodiments, the target sequence comprises a chemical modification to bind the probe to the polynucleotide. In some embodiments, the chemical modification is selected from the group consisting of acetylation, methylation, amorphization, glycosylation, phosphorylation, biotinylation, and oxidation.

Probe  molecule

In the art, probe molecules are detected or quantified by binding to a target-containing polynucleotide.

A probe used herein is understood to be capable of specifically binding to a certain site on a polynucleotide, which site is characterized by sequence or structure. Examples of probe molecules include PNA-conjugates that increase the size or charge of PNA (protein nucleic acid), bis-PNA, gamma-PNA, PNA. Other examples of probe molecules include natural or recombinant proteins, protein fusions, DNA binding domains of proteins, peptides, nucleic acids, oligonucleotides, TALEN, CRISPR, PNA (protein nucleic acid), bis-PNA, gamma-PNA, , Or a PNA-conjugate (e. G., Labeled oligo) that increases functionality, or any other PNA-derived polymer, and a compound.

In some embodiments, the probe comprises gamma-PNA. gamma -PNA has a simple modification in the peptide-like backbone, specifically at the gamma -position of the N- (2-aminoethyl) glycine backbone, thus generating a chiral center (Rapireddy S., et al. 1997, J. Am. Chem. Soc., 129: 15596-600; He G, et al., 2009, J. Am. Chem. Soc., 131: 12088-90; Chema V, et al., 2008, Chem. Soc., 128: 10258-10267). ≪ / RTI > Unlike bis-PNA, gamma-PNA can bind to dsDNA without sequence restriction, leaving one of the two DNA strands accessible to further hybridization.

In some embodiments, the function of the probe is to hybridize to a polynucleotide having a target sequence by a complementary base pair to form a stable complex. The PNA molecule is further coupled to additional molecules to form a complex with a sufficiently large cross-sectional surface area to provide a detectable change or contrast in signal amplitude beyond the background, which is the average signal amplitude corresponding to a section of the non-probe-bound polynucleotide .

The binding stability of the polynucleotide target sequence to the PNA molecule is important for detection by the nanopore device. Binding stability must be maintained over the period that the target-containing polynucleotide is displaced through the nanopore. If the stability is weak or unstable, the probe can be separated from the target polynucleotide and will not be detected because the target-containing polynucleotide can escape through the nanopore.

In certain embodiments, an example of a probe is a PNA-conjugate wherein a portion of the PNA specifically recognizes the nucleotide sequence and the portion of the conjugate increases the size / shape / charge difference between the different PNA-conjugates.

As illustrated in Figure 8, ligands A, B, C and D each specifically bind to a site on the DNA molecule, and these ligands can be identified by width, length, size and / or charge and distinguished from each other . When their corresponding sites are designated as A, B, C and D, respectively, confirmation of the ligand leads to the revelation of A-B-C-D in terms of the structure of the DNA sequence, site and sequence.

Different reactive moieties may be incorporated into the ligand to provide a chemical handle to which the label may be conjugated. Examples of reactive moieties include, but are not limited to, primary amines, carboxylic acids, ketones, amides, aldehydes, boronic acids, hydrazones, thiols, maleimides, alcohols, and hydroxyl groups, and biotin.

Figure 9A shows a modified PNA ligand to increase the ligand charge to facilitate detection by the nanopore. Specifically, this ligand, which binds to the target DNA sequence by a complementary base pair and a Hoogsteen base pairing between the base of the PNA molecule and the base of the target DNA, has a cysteine residue incorporated in the backbone, Lt; RTI ID = 0.0 > thiol < / RTI > Herein, cysteine is labeled via the maleimide linker to the peptide 2-aminoethylmethanethiosulfonate (MTSEA), which means that the label / peptide increases the charge of the ligand and therefore means for detecting whether the ligand binds to the target sequence . Larger charge results in a larger change in current flow through the pores compared to unlabeled PNAs.

In some embodiments, a pseudo-peptide is used to change the overall size of the ligand (e.g., PNA) to increase the contrast of the change between the ligand-bound polynucleotide and other background molecules present in the sample. Modifications to the backbone are made to increase contrast. For example, referring to FIG. 9B, this represents a PNA incorporating a cysteine residue 301 modified with SMCC linker 302 to enable conjugation to peptide 303 via the N-terminal amine of the peptide . In addition to the addition of charge through labeling of the ligand (e.g., as in Figure 9A), the selection of a more charged amino acid instead of a non-polar amino acid may serve to increase the charge of the PNA. In addition, small particles, molecules, proteins, peptides, or polymers (e.g., PEG) can be conjugated to the pseudo-peptide backbone to enhance the bulk or cross-sectional surface area of the ligand and target-containing polynucleotide complexes. The enhanced bulk serves to improve the signal amplitude contrast so that any differential signal resulting from the increased bulk can be easily detected. Examples of small particles, molecules, proteins, or peptides can be conjugated to a pseudo-peptide backbone and include alpha-helical peptides, nanometer (e. G., 3 nm) sized gold particles or rods, quantum dots, polyethylene glycol PEG). ≪ / RTI > Methods of conjugating molecules are well known in the art, for example, in U.S. Patent Nos. 5,180,816, 6,423,685, 6,706,252, 6,884,780, and 7,022,673, the disclosure of which is incorporated herein by reference .

While the above embodiments describe PEG labeling through cysteine residues, other residues may be used. For example, lysine residues are easily replaced with cysteine residues to enable linkage chemistry using NHS-esters and free amines. In addition, PEG can be easily replaced with other bulk-additive components, similar dendrons, beads, or rods between two functional linkers and PNAs, or can be coupled directly to a dendron. One of ordinary skill in the art will recognize the flexibility of the modified linkage chemistry for this particular system, for example serine-reactive isocyanate, in that the amino acid can be exchanged. Some examples of linking chemistries that can be used in this reaction are listed in the table below.

Connective Chemistry Reactive group Target functional group Aryl azide Non-selective or primary amine Carbodiimide Amine / carboxyl Hydrazide carbohydrate Hydroxymethylphosphine Amine Imido ester Amine Isocyanate Hydroxyl Carbonyl Hydrazine Maleimide Sulfhydryl NHS-ester Amine PFP-ester Amine Psoralen Thymine Pyridyl disulfide Sulphate Drill Vinyl sulphone Sulfhydryl amine, hydroxyl

Figures 3A, 3B, 4, 5, 9A, 9B, and 18A illustrate how to increase the size of the probe to facilitate detection or to detect if two probes are in proximity, to contain epitopes, to contain ssDNA oligomers, With the proviso that the PNA probe is modified to contain additional charge, or additional size.

Different reactive moieties may be incorporated into the probe to provide a chemical handle to which the label may be conjugated. Examples of reactive moieties include, but are not limited to, primary amines, carboxylic acids, ketones, amides, aldehydes, boronic acids, hydrazones, thiols, maleimides, alcohols, and hydroxyl groups, and biotin.

A common way to integrate chemical handles is to include specific amino acids in the backbone of the probe. Examples include, but are not limited to, cysteine (providing thiolate), lysine (providing free amine), threonine (providing hydroxyl), glutamate (glutamate) and aspartate (providing carboxylic acid).

Different types of labels may be added using reactive moieties. These include the following labels:

1. Labels which increase the size of the probe, for example biotin / streptavidin, peptides, nucleic acids

2. A label that changes the charge of the probe, e. G., A charged peptide (6xHIS), or a protein (e. G., Charybdotoxin), or a small molecule or peptide (e.

3. Labels that change the probe or add fluorescence to the probe, for example, general fluorophore, FITC, Rhodamine, Cy3, Cy5.

4. A label that provides an epitope or interaction site that binds to a third molecule, e. G., An antibody binding peptide.

Multiplexing

In some embodiments, instead of including the same kind of probe, a collection of different probes, each binding to a unique site or target motif, as described above, is added.

With this setting, a number of different probes can be used to detect a number of different target sites within the same target containing polynucleotide. Figure 10 shows this method. Herein, double-stranded DNA (1002) contains a number of different target motifs, two copies of 1003, two copies of 1004, and one copy of 1005.

By using probes 1006, 1007, and 1008, each providing a unique current profile (e.g., by varying the size), the technique can detect different target motifs in the same molecule, and target motif detection And provides means for multiplexing. In addition, the number of each target (or the number of copies) can be determined by listing how many of each unique probe are combined. By adjusting the conditions that affect the coupling, the system can obtain more detailed combined dynamic information.

Similarly, multiplexing can be achieved by having a collection of probes framed in any number of combinations with different attributes, and the only requirement is that the probes binding to the different sequences should be distinguishable from one another. For example, experiments can use size-distinguishable probes and additional probes that are distinguishable in size (Figures 9A, 9B, and 19).

Additional multiplexing methods involve designing probes that bind to the polynucleotides in known sequences at fixed locations of one another to obtain information in a sample containing a collection of nucleic acids of different species. As an example of this method, when we tested the source against three different bacteria of known sequence, we used two probes, 1000 bp apart for species A, 3000 bp for species B, It can be spaced apart by 5000 base pairs for species C. When probes 1000 base pairs and 3000 base pairs apart are detected, species A and B are present, but species C is not detectably present. Such a method of designing space can also be used for multiple detection of known or mutated sequences in a particular target sample.

Nanopore device

A nanopore device includes a pore that defines an opening in a structure that separates the interior space of the device into two volumes, as provided, for example, to identify an object passing through the pore using a sensor For example, by detecting a change in a parameter indicative of an object). The nanopore devices used in the methods described herein are also disclosed in PCT Publication WO / 2013/012881, the full text of which is incorporated herein by reference.

In a nanopore device, the pore (s) are nanoscale or microscale. In one embodiment, each pore has a size to pass small or large molecules or microorganisms. In one embodiment, each pore is at least about 1 nm in diameter. Alternatively, each pore may have a diameter of at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm to be.

In one embodiment, the pores are about 100 nm or less in diameter. Alternatively, the pore may have a diameter of about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, nm, 20 nm, 15 nm, or 10 nm or less.

In some embodiments, each pore has a diameter of at least about 100 nm, 200 nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm. In one embodiment, the pores are about 100,000 nm or less in diameter. Alternatively, the pores may have a diameter of about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, to be.

In one embodiment, the pore is at least about 1 nm to about 100 nm, or about 2 nm to about 80 nm, or about 3 nm to about 70 nm, or about 4 nm to about 60 nm, or about 5 nm to about 50 nm, Or from about 10 nm to about 40 nm, or from about 15 nm to about 30 nm.

In some embodiments, the pore (s) in the nanopore device are on a larger scale to detect large microorganisms or cells. In one embodiment, each pore has a size to pass large cells or microorganisms. In one embodiment, each pore is at least about 100 nm in diameter. Alternatively, each pore may have a diameter of at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, , 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm.

In one embodiment, the pore is about 100,000 nm or less in diameter. Alternatively, the pore may have a diameter of about 90,000 nm, 80,000 nm, 70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm, 10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, nm, 3000 nm, 2000 nm, or 1000 nm or less.

In one embodiment, the pores have a refractive index of from about 100 nm to about 10000 nm, or from about 200 nm to about 9000 nm, or from about 300 nm to about 8000 nm, or from about 400 nm to about 7000 nm, or from about 500 nm to about 6000 nm, Or from about 1000 nm to about 5000 nm, or from about 1500 nm to about 3000 nm.

In some embodiments, the nanopore device further comprises means for moving the polymer scaffold across the pore and / or means for identifying an object passing through the pore. A more detailed description is provided below and is described in the context of a two-for-device.

Compared to a single-fornone nanopore device, a two-forer device can be more easily configured to provide good control of the speed and direction of movement of the polymer scaffold across the pore.

In certain embodiments, the nanopore device includes a plurality of chambers, each chamber interchanging with an adjacent chamber through at least one pore. Of these pores, the two pores, the first pore and the second pore, are arranged such that at least a portion of the polymer scaffold moves out of the first pore and into the second pore. The device also includes a sensor that can identify the polymer scaffold on the move. In one embodiment, such confirmation entails identifying individual components of the polymer scaffold. In another embodiment, the confirmation comprises identifying the fusion molecule and / or the target analyte bound to the polymer scaffold. When a single sensor is used, a single sensor may include two electrodes disposed at both ends of the pore to measure the ion current across the pore. In yet another embodiment, a single sensor includes components other than electrodes.

In one aspect, the device includes three chambers connected through two pores. A device with three or more chambers can be easily designed to include one or more additional chambers on either side of the three-chamber device, or between any two of the three chambers. Similarly, two or more pores may be included in the device to connect the chambers.

In an embodiment, there may be more than one pore between two adjacent chambers such that multiple polymer scaffolds move simultaneously from one chamber to the next. This multi-pore design can improve the throughput of polymer scaffold analysis in the device.

In some embodiments, the device further comprises means for moving the polymer scaffold from one chamber to another. In one embodiment, this movement leads to loading of the polymer scaffolds simultaneously across both the first pore and the second pore. In another embodiment, the means also enable movement of the polymer scaffold in the same direction through the two pores.

For example, in a three-chamber two-fored device (a "two-pour" device), each chamber may have electrodes ≪ / RTI >

According to an embodiment of the present disclosure, there is provided a device comprising an upper chamber, an intermediate chamber and a lower chamber, wherein the upper chamber communicates with the intermediate chamber via a first pore, the intermediate chamber communicates with the lower chamber via the second pore, do. Such a device may have any of the dimensions or other features previously disclosed in U. S. Publication No. 2013-0233709 entitled " Dual-Pore Device ", which is incorporated herein by reference in its entirety.

In some embodiments, as shown in Figure 7A, the device includes an upper chamber 705 (chamber A), an intermediate chamber 704 (chamber B), and a lower chamber 703 (chamber C). The chambers are separated by two separation layers or membranes 701 and 702, each having a separate pore 711 or 712. In addition, each chamber contains electrodes 721, 722 or 723 for connection to a power supply. The tin in the upper, middle and lower chambers is a tin in the relative aspect, for example the upper chamber does not lie above the middle or lower chamber relative to the ground, or vice versa.

Each of the pores 711 and 712 independently has a size such that small or large molecules or microorganisms pass through it. In one embodiment, each pore is at least about 1 nm in diameter. Alternatively, each pore may have a diameter of at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, , 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, .

In one embodiment, the pores are about 100 nm or less in diameter. Alternatively, the pore may have a diameter of about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, nm, 20 nm, 15 nm, or 10 nm or less.

In one embodiment, the pore is at least about 1 nm to about 100 nm, or about 2 nm to about 80 nm, or about 3 nm to about 70 nm, or about 4 nm to about 60 nm, or about 5 nm to about 50 nm, Or from about 10 nm to about 40 nm, or from about 15 nm to about 30 nm.

In another embodiment, each pore has a diameter of at least about 100 nm, 200 nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm. In one embodiment, each pore has a diameter of 50,000 nm to 100,000 nm. In one embodiment, the pores are about 100,000 nm or less in diameter. Alternatively, the pores may have a diameter of about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, to be.

In some embodiments, the pores have a substantially rounded shape. The term "substantially round" as used herein refers to a form in which at least about 80 or 90% of the shapes are circular. In some embodiments, the pores are rectangular, rectangular, triangular, elliptical, or hexagonal in shape.

Each of the pores 711 and 712 independently has a depth (i.e., the length of the pore extending between two adjacent volumes). In one embodiment, each pore has a depth of at least about 0.3 nm. Alternatively, each pore may be at least about 0.6 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, nm. < / RTI >

In one embodiment, each pore has a depth of about 100 nm or less. Alternatively, the depth may be about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, , 20 nm, 15 nm, or 10 nm or less.

In one embodiment, the pore is at least about 1 nm to about 100 nm, or about 2 nm to about 80 nm, or about 3 nm to about 70 nm, or about 4 nm to about 60 nm, or about 5 nm to about 50 nm, Or from about 10 nm to about 40 nm, or from about 15 nm to about 30 nm.

In some embodiments, the nanopore extends through the membrane. For example, a pore may be a protein channel inserted into a lipid bilayer membrane or it may be a protein channel inserted into a lipid bilayer membrane or by a layer formed by drilling, etching, or a solid-state substrate such as silicon dioxide, silicon nitride, graphene, Lt; RTI ID = 0.0 > pores. ≪ / RTI > In some embodiments, the length or depth of the nanopore is large enough to form a channel connecting two separate volumes. In some embodiments, the depth of each pore is greater than 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. In some embodiments, the depth of each pore is 2000 nm or 1000 nm or less.

In one embodiment, the pores are spaced apart by a distance between about 10 nm and about 1000 nm. In some embodiments, the inter-fore distance is longer than 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, or 9000 nm. In some embodiments, the pores are spaced apart by 30000 nm, 20000 nm, or 10000 nm or less. In one embodiment, the distance is at least about 10 nm, or at least about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, Or 300 nm. In yet another embodiment, the distance is about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 150 nm, or 100 nm or less.

In another embodiment, the interforate distance is from about 20 nm to about 800 nm, from about 30 nm to about 700 nm, from about 40 nm to about 500 nm, or from about 50 nm to about 300 nm.

The two pores may be arranged at any position as long as the pores allow fluid flow between the chambers and have a defined size and distance between the pores. In one embodiment, the pores are arranged such that there is no direct interdiction between them. In one aspect, as shown in Figure 7A, the pores are substantially in the same axis.

In one aspect, as shown in FIG. 7A, the device is connected to one or more power supplies through electrodes 721, 722, and 723 of chambers 703, 704, and 705. In some aspects, the power supply includes a voltage-clamp or a patch-clamp, which can supply voltage across each pore and measure current independently through each pore. In this aspect, the power supply and the electrode configuration can set the intermediate chamber to a common ground for both power supplies. In one aspect, the power supply or supplies include a first voltage V ₁ between the upper chamber 705 (chamber A) and the intermediate chamber 704 (chamber B), and a first voltage V ₁ between the intermediate chamber 704 and the lower chamber 703 It is configured to apply a second voltage (V ₂₎ between (chamber C).

In some aspects, the first voltage (V ₁ ) and the second voltage (V ₂ ) are independently adjustable. In one embodiment, the intermediate chamber is adjusted to be ground for two voltages. In one embodiment, the intermediate chamber includes a medium for providing conductivity between each pore and an electrode of the intermediate chamber. In one embodiment, the intermediate chamber includes a medium for providing a resistance between each pore and an electrode of the intermediate chamber. Keeping this resistance small enough compared to the nanopore resistance is useful for isolating the two voltages and currents across the pores, which helps in independent adjustment of the voltage.

Adjustment of the voltage can be used to control the movement of the charged particles in the chamber. For example, when the two voltages are set to the same polarity, appropriately charged particles can be sequentially moved from the upper chamber to the intermediate chamber and back to the lower chamber, or vice versa. In some embodiments, when the two voltages are set to the opposite polarity, the charged particles may move to and remain in the intermediate chamber from the upper or lower chamber.

Adjustment of the voltage in the device can be particularly useful for controlling the movement of large molecules, e.g., charged polymer scaffolds, long enough to cross two pores simultaneously. In such an embodiment, the direction and speed of movement of the molecules can be controlled by the relative magnitude and polarity of the voltage as described below.

The device may contain a liquid sample, in particular a biological sample, and / or a material suitable for holding a material suitable for nanofabrication. In one embodiment, this material includes a dielectric material, such as silicon, silicon nitride, silicon dioxide, graphene, carbon nanotubes, TiO _2, HfO _2, Al ₂ O _3, or other metal layer, or any combination of these materials However, it is not limited thereto. In some embodiments, for example, a single sheet of about 0.3 nm thick graphene film can be used as the pore-containing film.

Devices that are microfluidic and contain two-pore microfluidic chip implementations can be fabricated by a variety of means and methods. For a microfluidic chip composed of two parallel membranes, the two membranes can be simultaneously drilled by a single beam to form two concentric pores, but in cooperation with any suitable alignment technique, different beams are used on each side of the membrane It is also possible to do. Generally, the housing ensures the sealed separation of chambers A-C. 7B, the housing provides minimal access resistance between the voltage electrodes 721, 722, and 723 and the nanopores 711 and 712 so that each voltage is, in principle, Lt; / RTI >

In one aspect, the device comprises a microfluidic chip (labeled as "dual-core chip") consisting of two parallel films connected by a spacer. Each membrane contains a pore drilled into a single beam through the center of the membrane. The device also preferably has a Teflon (R) housing for the chip. The housing ensures a sealed separation of chambers A-C and provides a minimum access resistance to the electrodes, ensuring that each voltage is in principle applied across each pore.

More specifically, the pore-containing film may be fabricated with a transmission electron microscope (TEM) grid having a silicon, silicon nitride, or silicon dioxide window of 5-100 nm thickness. The spacers may be formed using an insulator, such as SU-8, photoresist, PECVD oxide, ALD oxide, ALD alumina, or a deposited metal material such as Ag, Au, or Pt, Can be used to separate the membrane by occupying a small volume outside the aqueous portion. The holder is disposed in an aqueous bath which occupies the largest volume of chamber B. Chambers A and C are accessible by a larger diameter channel (less approach resistance) that causes film seals.

Focused electrons or ion beams can be used to naturally align the pores by drilling the pores through the membrane. The pores can also be made smaller in size by applying the correct beam focused on each layer. Any single nanofoam drilling method can also be used to drill pairs of pores in two membranes, taking into account the thickness of the membrane and possible drill depths for a given method. It is also possible to further improve the film thickness by pre-drilling the micro-pores to a predetermined depth and then pre-drilling the nanopores through the remainder of the film.

In another embodiment, the insertion of biological nanopores into a solid-state nanopore to form a hybrid pore can be used in one or two pores in a two-pore method. Biological pores can increase the sensitivity of ionic current measurements and are useful, for example, for sequencing, when only single-stranded polynucleotides need to be captured and controlled in a two-for-all device.

By the voltage present in the pores of the device, the charged molecules can be moved through the pores between the chambers. The moving speed and direction can be controlled by the magnitude and polarity of the voltage. Also, since each of the two voltages can be independently adjusted, the direction and speed of movement of charged molecules can be precisely controlled in each chamber.

One example relates to a charged polymer scaffold, such as DNA, having a length that is longer than the combined distance comprising the depth of the two pores and the distance between the two pores. For example, the length by dsDNA is about 340 nm, which is substantially longer than 40 nm across two 10 nm-deep pores isolated at 20 nm. In the first step, the polynucleotide is loaded into the upper or lower chamber. By negative charge under physiological conditions at a pH of about 7.4, the polynucleotide can be moved across the pores to which the voltage is applied. Thus, in the second step, two voltages are applied to the pores at the same polarity and on the same or similar scale to sequentially move the polynucleotides across the two pores.

By the time the polynucleotide reaches the second pore, one or both of the voltages can be changed. Since the distance between the two pores is chosen to be shorter than the length of the polynucleotide, when the polynucleotide reaches the second pore, it is also in the first pore. Thus, as shown in Figure 7C, a rapid change in the voltage polarity in the first pore will generate a force pulling the polynucleotide from the second pore.

Two forks have the same voltage-force effect | V ₁ | = | V ₂ | +? V , the value? V > 0 (or < 0) Can be adjusted for the adjustable movement in the direction V ₁ (or V ₂ ). In practice, the voltage-induced forces in each pore will not be equal to V ₁ = V ₂ , but calibration experiments can identify appropriate bias voltages that produce the same attraction for a given two-fored chip ; A change around the bias voltage can be used for direction control.

If the magnitude of the voltage-induced force in the first pore is less than the voltage-induced force in the second pore, the polynucleotide will continue to cross the two pores at a slow rate toward the second pore. From this viewpoint, it can be seen that the speed and direction of movement of the polynucleotide can be controlled by the polarity and magnitude of the two voltages. As will be described further below, this sophisticated control of movement applies extensively.

Thus, in one aspect, a method of controlling movement of a charged polymer scaffold through a nanopore device is provided. The method comprises the steps of: (a) loading a sample comprising a charged polymer scaffold in one of the upper chamber, intermediate chamber, or lower chamber of any of the above embodiments, 1 voltage, and one or more power supply devices providing a second voltage between the intermediate chamber and the lower chamber; (b) setting an initial first voltage and an initial second voltage to position the polymer scaffold across both the first and second pores by moving the polymer scaffold between chambers; And (c) adjusting a first voltage and a second voltage such that the first voltage and the second voltage from the intermediate chamber generate a force to attract the charged polymer scaffold, Scale under controlled conditions, involves moving the charged polymer scaffold across the two pores in both directions and in a controlled manner.

To demonstrate the voltage-competition approach in step (c), the relative force exerted by each voltage in each pore should be determined for each of the two-forearn devices used, which may be different for the motion of the polynucleotide Can be carried out by a calibration experiment by observation of the influence of the voltage value, which can be measured by detecting known positions and detectable characteristics at the polynucleotide, examples of which are described in more detail below in this disclosure. If the forces are equal at each common voltage, for example, the use of the same voltage value at each pore (having a common polarity in the upper and lower chambers relative to the grounded intermediate chamber) results in zero net motion zero net motion) (the presence and effect of Brownian motion is discussed below). If the forces are not equal at each common voltage, achieving the same force involves the identification and use of a larger voltage in the pores experiencing a weaker force at the common voltage. Calibration of the voltage-competition scheme may be performed for each two-fored device and for a particular charged polymer or molecule having characteristics that affect force when passing through each pore.

In one aspect, a sample containing a charged polymer scaffold is loaded into an upper chamber, and an initial first voltage is set to draw a charged polymer scaffold from the upper chamber to the intermediate chamber, And is configured to pull the fold from the intermediate chamber to the lower chamber. Similarly, the sample may initially be loaded into the lower chamber, and the charged polymer scaffold may be drawn into the intermediate chamber and the upper chamber.

In another aspect, a sample containing a charged polymer scaffold is loaded into an intermediate chamber, and an initial first voltage is set to draw the charged polymer scaffold from the intermediate chamber to the upper chamber, Lt; RTI ID = 0.0 &gt; polymer &lt; / RTI &gt; scaffold from the intermediate chamber to the lower chamber.

In one embodiment, the adjusted first and second voltages in step (c) are on the order of about 10 to about 10,000 times larger / different than the difference between the two voltages. For example, the two voltages may be 90 mV and 100 mV, respectively. The magnitude of the two voltages, about 100 mV, is about 10 times the difference / differential, 10 mV, between them. In some embodiments, the magnitude of the voltage is at least about 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 400 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times or 9000 times. In some embodiments, the magnitude of the voltage is about 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2000 times, 1000 times, 500 times, 400 times , 300 times, 200 times, or 100 times or less.

In one embodiment, the real-time or on-line adjustment to the first voltage and the second voltage in step (c) may be performed at a clock rate of up to several hundred megahertz, by active control or feedback control using dedicated hardware and software . The automated control of the first, second, or both voltages is based on feedback of the first, second, or both ion current measurements.

sensor

In certain embodiments, the nanopore device of the present invention includes one or more sensors to perform confirmation of the binding status of the target motif.

The sensor used in the device may be any sensor suitable for identifying molecules or particles, e.g., charged polymers. For example, the sensor can be configured to identify the charged polymer by measuring current, voltage, pH, optical characteristics associated with the charged polymer, or residence time or one or more individual components of the charged polymer. In some embodiments, the sensor is configured to measure the ion current passing through the pores as molecules or particles, particularly charged polymers (e.g., polymeric nucleolites) travel through the pores, Lt; / RTI &gt;

In certain embodiments, the sensor measures the optical characteristics of the polymer (or unit) of the polymer or polymer. One example of such a measurement includes confirmation by infrared (or ultraviolet) spectroscopy of a specific unit-specific absorption band.

When a residence time measurement is used, the residence time will be related to the size of the unit for a particular unit based on the length of time it took to pass through the sensing device.

In some embodiments, the sensor is functionalized with a reagent that forms a characteristic non-covalent bond with each probe. In this regard, the gap can allow larger and more effective measurements. For example, a 5 nm gap can be used to detect probe / target complexes measuring approximately 5 nm. Tunnel sensing using a functionalized sensor is called "recognition tunneling ". Using a scanning tunneling microscope (STM) with recognition tunneling, probes coupled to the target motif are readily identified.

Thus, the methods of the present technology can control the delivery rate of charged polynucleotides (e. G., DNA) to one or more recognition tunneling sites, each located in one or two nanopore channels or between the pores, Control can ensure that each probe / target complex is at each site for a sufficient period of time to be validated.

The sensor of the device and the method of the present disclosure may comprise gold, platinum, graphene, or carbon, or other suitable material. In certain embodiments, the sensor includes a component made of graphene. Since graphene can act as a conductor and an insulator, the tunneling current through the graphene and across the nanopore can sequence the potential DNA.

In some embodiments, the tunnel gap has a width from about 1 nm to about 20 nm. In one embodiment, the width of the gap is at least about 1 nm, or at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12 or 15 nm. In another embodiment, the width of the gap is about 20 nm or less, or about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, Or 2 nm or less. In some embodiments, the width is from about 1 nm to about 15 nm, from about 1 nm to about 10 nm, from about 2 nm to about 10 nm, from about 2.5 nm to about 10 nm, or from about 2.5 nm to about 5 nm.

In some embodiments, the sensor is an electrical sensor. In some embodiments, the sensor detects fluorescence detection means when the probe has a label that produces a unique fluorescence signature. A radiation source at the outlet can be used to detect the signature.

While the invention will be described in conjunction with the foregoing embodiments, it is to be understood that the foregoing description and the following examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the present invention will become apparent to those skilled in the art to which the present invention belongs.

Example

Example  1 - solid state Nanopore  In the experiment, DNA alone

The nanopore mechanism uses a sensitive voltage-clamp amplifier to apply a voltage V across the pore while measuring the ion current (I ₀ ) through an open pore. Single charged molecules, such as double-stranded DNA (dsDNA) is captured by electrophoresis when driven through the pore, the measured current is moved from I ₀ to I _B and, ΔI = I ₀ - I _B and the period t _D was used to characterize the event. After recording a number of events during the experiment, the distribution of events in the ΔI vs. t _D plot is analyzed to characterize the molecule in the population of plots. In this way, nanopores provide a simple, label-free, purely electrical single-molecule method for biomolecule sensing.

A single nanopore fabricated on silicon nitride (SiN) substrate appears as an example of a solid-state nanopore with a 40 nm diameter pore in a 100 nm thick SiN film (FIG. 11A). In Figure 11B, a typical current tracing is a blocking event triggered by a 5.6 kb dsDNA that passes through a 11-nm diameter nanopore of 10 nm thick SiN at 200 mV in a buffer containing 1 M KCl in a single file format (unfolded) . The current increases as the DNA passes through the pore at a KCl concentration of less than 0.3 M, whereas DNA decreases when the DNA passes through the pore at a KCl concentration of 0.3 M or more. The average open channel current is I ₀ = 9.6 nA, the average event amplitude is I _B = 9.1 nA, and the average event duration is t _D = 0.064 ms. The amplitude shift of the dsDNA molecule through the nanopore is ΔI = I ₀ - I _B = 0.5 nA. In Fig. 11C, the scatter plot shows |? I | for all 1301 events recorded for 16 minutes. To t _D.

Example  2-containing PNA and biotin for target sequence detection capture  molecule

The present inventors have demonstrated a method of allowing the detection of binding of a compound in solution by a manipulated polymer scaffold. The present inventors provide a PNA probe modified to contain a biotin moiety that binds to a neutravidin. Nutrabydines increase the bulk, making the PNA detectable in large nano-pores (eg, 15-30 nm diameter). Specifically, we engineered a 5.6 kb dsDNA scaffold to bind to a 12-mer peptide-nucleic acid (PNA) probe molecule, each PNA probe having three biotinylated sites that are each linked to a neurotavidin 12A). We engineered the dsDNA scaffold to have 25 distinct sites (binding motifs) that bind to PNA probes (Figure 12B). We provided a solution comprising a polymer scaffold alone, free neutravidin, or a probe / DNA complex (Figure 13). Current event signatures obtained from each group lead to a detectable potential signatures that are more detectable than other background event types (eg, unbound DNA alone, neutravidin alone, PNA / neutravidin alone), DNA / PNA / Indicating that it can be confirmed in the nanopore (FIG. 13). In this example, we show that DNA / PNA / Nutravidin complexes can be detected with high confidence in nanopores.

A probe that binds to a specific DNA sequence is a protein nucleic acid molecule (PNA) that binds to a unique sequence (GAAAGTGAAAGT, uSeql) repeated 25 times throughout the scaffold. The PNA used in the experiment has the sequence GAA * AGT * GAA * AGT, where * is incorporated into the PNA backbone at the gamma position by coupling to biotin with lysine amino acids, so that each PNA has three biotin molecules and potentially (PNABio), which binds to three neu- trastine molecules. To bind PNA, a 60 nM scaffold was heated to 95 [deg.] C for 2 min, cooled to 60 [deg.] C and incubated with PNA in excess of 10x for possible PNA- binding sites on the scaffold for 1 hour at 15 mM NaCl Gt; 4 C &lt; / RTI &gt; Excess PNA was dialyzed against 10 mM Tris pH 8.0 for 2 hours (20k MWCO, Thermo Scientific). This DNA / PNA complex is then labeled with a 10-fold excess of neutavidin protein (Pierce / Thermo Scientific) on possible biotin sites bound to the scaffold (assuming a 60% reduction in PNA during dialysis). Reactions are electrophoresed as described above to assess purity, concentration, and potential aggregation.

Figure 14A-B shows the data for comparing the ΔI for t _D dispersion from the following three separate experiments: DNA alone (D), Nutra neutravidin alone (N), and D / P / N reagent (DPN). In the D / P / N experiment, the largest | ΔI | The event is due to the D / P / N complex (Figure 13), which is based on the binding state (i.e., unbound, scaffold with PNA, and scaffold with PNA and combined neutravidin) Provides a simple criterion for. Specifically, the present inventors have found that |? I | > 4nA, it is possible to display the event corresponding to the D / P / N complex. For the data set of FIG. 14A, only 9.4% (390) of the D / P / N experiments were deviated by |? I |> 4nA whereas only 0.46% of D and 0.16% of N events in the control group exceeded 4 nA do. In a separate experiment (data not shown) with 1 M KCl and an applied 200 mV 7 nm diameter pore, no events exceeded 4 nA (0%) in the control group with PNA and neutravidin at 0.4 nM concentration . Applying our mathematical criterion, the random variable Q = {part of the marked event} has a binomial distribution, and using these and other statistical modeling tools, the inventors have found that for this data set to Q = 9.29 + 1.15% 99% confidence interval can be calculated. Since 9.29%> 0.46% (maximum false positive%) is fully satisfied within the 99% confidence interval for Q, the inventors have positive test results and collect data within 8 minutes. In fact, the same 99% confidence is achieved with only the first 60 seconds of data for this data set. Gel migration indicates that scaffold DNA transfer is delayed in a neu- trambin-dependent manner (Fig. 14C); This result leads us to use the 10x concentration in this preliminary experiment, since all the DNA is labeled and a nearly homogeneous population appears to be produced.

Example  3 - DNA On the scaffold  Combine VspR  Protein and Nanopore  detection

The VspR protein is a 90 kDa protein of V. cholerae that binds directly to dsDNA with high micromolar affinity in a sequence-specific manner (Yildiz, Fitnat H., Nadia A. Dolganov, and Gary K. Journal of bacteriology 183, no. 5 (2001), " Vectors of Vibrio cholerae O1 El Tor. " : 1716-1726). In this example of target sequence detection using nanopore technology, VspR serves as a probe molecule with a site-specific DNA binding domain. In this experiment, the present inventors confirmed the detection of VspR on a DNA scaffold as a model using a protein to detect the presence of a specific sequence in DNA. The DNA scaffold contained 10 VspR specific binding sites (Figure 15). To preserve the affinity of VspR for dsDNA binding, we used 0.1 M KCl, the salt concentration at which the VspR-bound scaffold dislocations through the nanopore improve the current flow through the nanopore (Figure 16) . We provided a solution containing the VspR protein at a concentration of 18 nM in the recording buffer and at a concentration of 180 nM during labeling (binding step). This results in an excess of 18x the VspR protein on the DNA binding site. Experiments were performed at pH 8.0 (pI of VspR protein was 5.8). Considering Kd and DNA concentration, only 0.1-1% of the DNA should be completely occupied by VspR, partly occupying a higher percentage, and some unrecognized remaining percentages of DNA are not totally bound. In addition, the free VspR protein is present in the solution during the nanopore experiment.

Two representative events are shown in Figures 16A and 16B. In the experiment using VspR, the VspR concentration was 18 nM (1.6 mg / L), 10 nM binding site. The scaffold concentration was 1 nM, resulting in capture every 6.6 seconds. Pore size is 15 nm in diameter and length. The voltage is -100 mV and the negative voltage produces a negative current so that the upshift corresponds to an attenuation event as shown for the VspR-associated DNA event (Figure 16B), while the downshift corresponds to the unbound DNA scaffold To generate a positive shift as shown in the event (Figure 16A). This corresponds to the idealized signal pattern and condition in FIG. 2, and the DNA event (FIG. 16A) has a faster period and opposite polarity compared to the fusion molecule-bound DNA event (FIG. 16B). Thus, an important observation in this figure shows that the presence of a specific DNA sequence is readily detectable since the VspR-linked event has opposite signal polarity compared to unbound DNA events. Figure 17 shows at least ten representative current decay events consistent with the VspR-coupled scaffold through the pores. There are 90 such events for 10 minutes to record, which corresponds to one VspR-combined event per 6.6 seconds. The event was attenuation with an amplitude of 50 to 150 pA and a duration of 0.2 to 2 milliseconds. 16-17, the downward event corresponds to the current enhancement event, the upward event corresponds to the current attenuation event, and even if the baseline is set to zero for display purposes, this direction of movement is preserved.

Example  4 - Sequence specific Probe  Synthesis and Target Sequence Binding

In this example, we show the production of a PNA probe for binding to the desired target sequence, and the features added to the PNA allow for increased sensitivity of detection in the nanopore.

The present inventors have produced bisPNA probes containing three cysteine residues. The bisPNA probe comprises a sequence of a PNA capable of binding to a DNA sequence comprising the target sequence of CTTTCCC at the position of this target sequence on the target DNA molecule. The bisPNA probes were also labeled with maleimido-PEG-Me in the three cysteine residues on the bisPNA probe to increase the detection of probes attached to the target DNA molecule in the nanopore. PEG-CEG-Cys-Lys-Lys) with a bispNA (Lys-Lys-Cys-PEG3-JTTTJJJ-PEG-Cys-PEG-CCCTTTC-PEG-Cys-Lys-Lys) under reducing conditions with a 100-fold excess of linker &Lt; / RTI &gt; and incubated together to generate a PNA-PEG probe. The maleimide moiety of the linker reacts with the free thiol of PNA at pH 7.4, thus producing PEGylated-PNA. The addition of lysine increases the reagent affinity for specific homologous DNA sequences, allowing them to remain bound under high salt conditions (1 M LiCl). The PNA-PEG probe bound to the target sequence on the dsDNA molecule is shown in Figure 18A.

To confirm the binding of the DNA-PEG probe to the target sequence in the DNA molecule, we performed gel migration assays using solutions obtained by incubating different versions of the PEG probe with DNA. For this assay, we used four samples as shown in Figure 18B. Lane 1 is DNA alone, lane 2 is DNA + PNA, lane 3 is DNA + PNA-PEG (10 kDa), and lane 4 is DNA + PNA-PEG (20 kDa). The uptake in lanes 2-4 is consistent with the bisPNA species bound to DNA. The prototype is DNA / PNA-PEG and the boxed species is DNA / PNA of lanes 3 and 4 present as residual PNA (without PEG) in labeling experiments. The results of the gel migration assay indicate the complex formation of PNA probes with homologous DNA sequences complementary to the target sequence, regardless of the attachment of PEG to the DNA containing the target sequence and PNA. Thus, we have identified successful complex formation of sequence-specific probes that can be detected in nanopores.

The inventors then performed assays to show the specificity of the PNA probes for the target DNA sequence. In this application, the present inventors used a PNA probe (without PEG) as a template, a DNA with no target sequence (lane 2), a DNA with a target sequence containing a single base mismatch with a PNA probe (lane 3) (Lane 4), and each sample was analyzed using a gel migration assay, the results of which are shown in Figure 18C. Lane 1 is a DNA marker. As shown in this result, DNA (lane 4) having the target sequence correctly binds to PNA while DNA (target 3) having the target sequence containing the single base mismatch sequence (lane 3) and DNA without the target sequence Random sequence (instead of the target sequence) (lane 2) does not exhibit PNA binding. Therefore, gel migration assays indicate that PNA specifically binds to DNA containing the target sequence and does not bind even DNA with a single mismatch in the target sequence.

Example  5-Modified sequence-specific The probe  using In the nanopore  Target sequence detection

In this example, we show detection of a DNA molecule comprising a target sequence bound to a PEG-modified sequence-specific PNA probe.

The present inventors have provided three different PNA probes to have different bulk properties based on PEG attachment and PEG length. Three types of probes were used: 1) PNA without PEG, 2) PNA coupled to 5 kDa PEG, and 3) PNA coupled to 10 kDa PEG. Each probe was mixed with DNA containing the target sequence and DNA bound to the PNA probe was detected in the nanopore. The concentration of each complex in the sample was 2 nM in 1 M LiCl buffer. The sample was run under an applied voltage of 100 mV in a nanopore device. The results are shown in Figures 19A-19E.

In Figure 19A, the representative individual events observed are for DNA bound to each type of probe. The event signature for the DNA / bisPNA event appears on the left. The event signatures of DNA / bisPNA-PEG complexes with a maximum of 3 PEGs bound to each PNA and a 5 kDa PEG size are intermediate. The event signatures of the DNA / bisPNA-PEG complex with up to 3 PEGs bound to each PNA and 10 kDa PEG are shown on the right. The event signatures for each were measured by current interruption through the nanopores in the potential of the identified complexes. In Figure 19A, the molecular diagram represents linearized PEG and DNA scaled for visual comparison. As the probe size (bulk) increases, the event signature changes.

The inventors analyzed the event group and created a scatter plot of the mean conductivity shift (dG) versus time for all events in each data set. We have generated scatterplots of event mean conductivities (mean current shifts divided by voltage) versus event durations (widths) from experiments as shown in Figure 19B. Plots show that DNA / PNA, DNA / PNA-PEG (5 kDa), and DNA / PNA-PEG (10 kDa) provide distinct overlapping groups based on event duration and average conductivity. We generated a histogram to represent the observed differences in mean conductivity shifts (dG) for each event between different complexes (Figure 19C). We also generated a histogram to represent the observed differences in event duration between different complexes (Figure 19D).

Example  6 - In the nanopore  To detect human cystic fibrosis Mutated  Detection of cftr gene target sequence

The present inventors confirmed the specificity of the binding of the modified PNA to the target sequence and the ability to detect the target sequence using the probe in the nanopore device. The present inventors have considered the use of a modified PNA probe in a nanopore device to detect a mutation-inducing disease, in particular a cystic fibrosis, in a sample of a patient.

We generated a modified PNA probe (PNA-PEG probe) comprising a PNA molecule that specifically binds to a target DNA sequence comprising a cftr gene with a mutation (? F508) that induces cystic fibrosis 4). &Lt; / RTI &gt; The PEG bound to the PNA probe was 5 kDa. DNA containing cystic fibrosis disease mutations was incubated with PEGylated PNA specific for mutation. The samples were then placed on a nanopore device with a 26 nm pore and potential events via nanopores were recorded and analyzed.

The potential event signature associated with the potential of the PNA-PEG probe bound to the DNA molecule was observed in a sample with DNA containing cystic fibrosis causing a mutation (ΔF508). Representative event signatures are shown in Figure 20A. Experiments using samples with DNA alone or with DNA / PNA alone (i.e., no PEG-PNA) do not provide a higher potential event than background, including the ability of the pore to accurately identify PNA-PEG probes bound to DNA, and Indicating improved detection by the modified probe provided herein. For the event set recorded from the sample with the target mutant gene and the PNA-PEG probe, the event was characterized by average conductivity shift and duration. Figure 20B shows the average conductivity shift versus period plot for each recorded event. 20C and 20D show corresponding histograms for characterizing the events detected by the average conductivity shift and duration of each event. The analyzed data corresponded to the expected data for the DNA / PNA-PEG (5 kDa) complex potential via nanopores, indicating successful binding and identification of the cftr mutant target sequence in the nanopore device.

We also tested PNA-PEG (5 kDa) specific for the ΔF508 cftr gene mutation with a sample containing 300 bp DNA with wild-type cftr sequence (lane 2) and a sample containing 300 bp DNA with ΔF508 cftr mutation (lane 3) ) Probe was carried out (Figure 20E). This data shows that the PNA-PEG probe specifically binds only the? F508 target sequence, but not the wild-type sequence.

Therefore, the inventors have successfully detected DNA comprising a single base cftr gene mutation (? F508) and have found that the use of our system to detect specific sequences of multiple nucleic acids in a sample, including diagnostic or therapeutic indication of a human patient .

Example  7 - In the nanopore  PNA-PEG The probe  Detection of infectious bacteria used

In this example, we have identified the use of a modified probe to detect the presence of bacterial DNA in a sample using a nanopore device.

The present inventors have synthesized probes using PNA molecules capable of specifically binding to Staphylococcus mitis bacterial DNA. bisPNA contains a sequence complementary to a sequence specific to S. mitis bacterial species.

In this assay, PNA probes are bound to 10 kDa PEG so that detection is allowed in the nanopore when bound to bacterial DNA. We observed binding by mixing PNA probes with bacterial DNA and performing gel migration assays in the samples. Figure 21A shows the results of a gel migration assay in which lane 1 contains bacterial DNA without a PNA probe and lane 2 contains bacterial DNA with a PNA probe. Observations show that PNA / PEG (10 kDa) probe is bound to S. mitis bacterial DNA.

The inventors then prepared two samples for detection in the nanopore. The first sample contained bacterial DNA (DNA / bisPNA-PEG) with a PEG-modified PNA probe. The second sample contained bacterial DNA alone. We run these samples through the nanopore device in two successive experiments and analyzed the resulting events. Figure 21B shows a scatter plot of the mean conductivity shift (dG) on the ordinate vs. the duration on the abscissa for all events recorded in two subsequent experiments. An event featuring tagged sample 1 (rectangle) and untagged sample 2 (circle) appears.

The tagged molecule is consistently higher than the background titer (dotted line), while the untagged molecule is lower than the dotted line and coincides with the background population. Molecular clusters of various background experiments (PEG-free DNA / PNA, filtered serum, etc.) were used to establish potency to display tagged events. Background events are not shown here. For accurate detection of bacterial DNA in the sample, the DNA must be tagged using highly site-specific probes.

These results show that the PNA / PEG-linked population of S. mitis bacterial DNA can be identified from background events, but not DNA alone or DNA / PNA alone. Thus, the modified PNA-PEG sequence-specific probe enables reliable detection of the presence or absence of S. mitis DNA in the sample.

Other Concrete example

It is to be understood that the words used are words of description rather than limitation and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

While the present invention has been described in terms of certain lengths and with a certain degree of specificity for the various described embodiments, it is not intended to be limited to any particular matter or embodiment or any particular embodiment thereof, Should be interpreted with reference to the appended claims to effectively incorporate the intended scope of the invention by providing a broad and possible interpretation of the scope of such claims.

All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of dispute, the present specification including definitions will be adjusted. In addition, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

<110> TWO PORE GUYS, INC. <120> TARGET SEQUENCE DETECTION BY NANOPORE SENSING OF SYNTHETIC PROBES <130> IF17P069US <150> US 62 / 056,378 <151> 2014-09-26 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 1 gaaagtgaaa gt 12 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 2 His His His His His   1 5

Claims (39)

A method for detecting a polynucleotide comprising a target sequence in a sample,
a) contacting the sample with a probe that specifically binds to the polynucleotide comprising the target sequence under conditions that promote binding of the probe to the target sequence to form a polynucleotide-probe complex;
b) loading the sample into a first chamber of a nanopore device, wherein the nanopore device includes at least one nanopore and at least the first chamber and the second chamber, Wherein the nanopore device is electrically and fluidly interchanged via the at least one nanofoer, wherein the nanopore device further comprises a voltage that is independently controlled across each of the at least one nanofoore and a sensor associated with each of the at least one nanofoore Wherein the sensor is configured to identify an object passing through at least one nanopore, the potential of the polynucleotide-probe complex through the at least one nanopore being determined by a detectable signal associated with the polynucleotide-probe complex ; And
c) observing the detectable signal to determine the presence or absence of the polynucleotide-probe complex in the sample by detecting the polynucleotide comprising the target sequence.
2. The method of claim 1, wherein the polynucleotide is DNA or RNA.
2. The method of claim 1, wherein the detectable signal is an electrical signal.
The method of claim 1, wherein the detectable signal is an optical signal.
2. The method of claim 1, wherein the probe comprises a molecule selected from the group consisting of a protein, a peptide, a nucleic acid, a TALEN, a CRISPR, a peptide nucleic acid, or a compound.
The probe of claim 1, wherein the probe comprises a molecule selected from the group consisting of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), DNA / RNA hybrid, polypeptide, or chemically derived polymer . &Lt; / RTI &gt;
2. The method of claim 1, wherein the probe comprises a PNA molecule coupled to a second molecule configured to facilitate detection of a probe bound to the polynucleotide in a potential across the at least one nanopore.
8. The method of claim 7, wherein the second molecule is PEG.
9. The method of claim 8, wherein the PEG has a molecular weight of at least 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, or 10 kDa.
2. The method of claim 1, further comprising applying to the sample a condition suspected of altering the binding interaction between the probe and the target sequence.
11. The method of claim 10, wherein the condition is selected from the group consisting of removal of the probe in the sample, addition of an agonist that competes with the probe for binding to the target sequence, and a change in initial pH, salt, or temperature condition Way.
2. The method of claim 1, wherein the polynucleotide comprises a chemical modification configured to modify the binding of the polynucleotide to the probe.
13. The method of claim 12, wherein the chemical modification is selected from the group consisting of biotinylation, acetylation, methylation, summation, glycosylation, phosphorylation, and oxidation.
2. The method of claim 1, wherein the probe comprises a chemical modification coupled to the probe via a cleavable bond.
The method of claim 1, wherein the probe interacts with a target sequence of the polynucleotide through a covalent bond, a hydrogen bond, an ionic bond, a metal bond, a van der Waals force, a hydrophobic interaction, or a planar stacking interaction Way.
2. The method of claim 1, further comprising contacting the sample with at least one detectable label capable of binding to the probe or polynucleotide-probe complex.
2. The method of claim 1, wherein the polynucleotide comprises at least two target sequences.
2. The method of claim 1, wherein the nanopores are from about 1 nm to about 100 nm in diameter and from about 1 nm to about 100 nm in length, and each chamber comprises an electrode.
2. The method of claim 1, wherein the nanopore device comprises at least two nanopores configured to simultaneously control the movement of the polynucleotide in all nanopores.
2. The method of claim 1, wherein the initial detection of the polynucleotide-probe complex by the detectable signal after the probe-binding portion has passed through the nanopore, Reversing the voltage, thereby again confirming the presence or absence of the polynucleotide-probe complex.
2. The method of claim 1, wherein the nanopore device comprises two nanopores, wherein the polynucleotides are simultaneously located in both nanopores.
22. The method of claim 21, further comprising adjusting the magnitude and / or direction of the voltage at each of the two nanopoles such that an opposing force is generated by the nanopore to control the rate of the polynucleotide's potential through the nanopore . &Lt; / RTI &gt;
A method for detecting a polynucleotide or polynucleotide sequence in a sample,
the method comprising: a) contacting the sample with a first probe and a second probe, wherein the first probe is capable of binding to the first target sequence of the polynucleotide under conditions that promote binding of the first probe to the first target sequence The second probe specifically binding to a second target sequence of the polynucleotide under conditions that promote binding of the second probe to the second target sequence;
b) combining said sample with said first and second probes simultaneously if said first and second probes are sufficiently close to each other under conditions that promote binding of said third molecule to said first probe and said second probe; To form a fusion complex comprising the polynucleotide, the first probe, the second probe, and the third molecule;
c) loading the sample into a first chamber of a nanopore device, wherein the nanopore device includes at least one nanopore and at least the first chamber and the second chamber, Wherein the nanopore device further comprises a sensor associated with each of the at least one nanofourous and a voltage potential controlled across each of the at least one nanofoure, wherein the nanopore device is electrically and fluidly interchanged through the at least one nanofoure, Wherein the sensor is configured to identify an object passing through the at least one nanopore, the fusion potential through the at least one nanopore providing a detectable signal associated with the fusion complex; And
d) observing the detectable signal to determine the presence or absence of the fusion complex in the sample.
24. The method of claim 23, wherein the polynucleotide is DNA or RNA.
24. The method of claim 23, wherein the detectable signal is an electrical signal.
24. The method of claim 23, wherein the detectable signal is an optical signal.
24. The method of claim 23, wherein the sufficient proximity is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, , 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or less than 500 nucleotides.
24. The method of claim 23, wherein the third molecule comprises PEG or an antibody.
24. The method of claim 23, wherein the third molecule and the first and second probes are bound to ssDNA and the ssDNA linked to the third molecule comprises a region complementary to a region of ssDNA linked to the first probe, Lt; RTI ID = 0.0 &gt; ssDNA &lt; / RTI &gt; linked to the second probe.
24. The method of claim 23, further comprising contacting the sample with one or more detectable labels capable of binding to a third molecule or fusion complex.
A kit comprising a first probe, a second probe, and a third molecule, wherein the first probe is configured to bind to a first target sequence on the target polynucleotide, and the second probe is configured to bind to a second target sequence on the target polynucleotide Wherein the third molecule is configured to bind to a first probe and a second probe when the first and second probes are bound to the polynucleotide in the first and second target sequences, Wherein the first and second probes are positioned sufficiently close to allow simultaneous binding of the third molecule to the first and second probes.
32. The kit of claim 31, wherein the first probe and the second probe are selected from the group consisting of a protein, a peptide, a nucleic acid, a TALEN, a CRISPR, a peptide nucleic acid, or a compound.
32. The kit of claim 31, wherein said third molecule comprises PEG or an antibody.
32. The kit of claim 31, wherein the third molecule comprises a modification to alter binding affinity for the probe.
A nanopore device comprising at least two chambers and nanopores, the device comprising a modified PNA probe coupled to a polynucleotide in the nanopore.
A dual-pore, dual-amplifier device for detecting a charged polymer through two pores, the device comprising an upper chamber, an intermediate chamber and a lower chamber, wherein the first pore connects the upper chamber and the intermediate chamber, Wherein the pore connects the intermediate chamber and the lower chamber, and wherein the device comprises a modified PNA probe coupled to the polynucleotide in the first or second pore.
37. The device of claim 36, wherein the device is configured to simultaneously control movement of the charged polymer through both the first pore and the second pore.
37. The device of claim 36, wherein the modified PNA probe is bound to at least one PEG molecule.
37. The apparatus of claim 36, wherein the device further comprises a power supply configured to provide a first voltage between the upper chamber and the intermediate chamber and to provide a second voltage between the intermediate chamber and the lower chamber, The intermediate chamber is connected to a common ground for two voltages and the device provides a dual-amplifier electronics configured for independent voltage control and current measurement in each pore, and the two voltages are different in magnitude And wherein the first and second pores are configured such that the charged polymer can move simultaneously across both pores in both directions and in a controlled manner.

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